A variety of immunodiagnostic assays are known to persons of ordinary skill in the art. For example, in the competitive solid phase immunoassay architecture, labeled tracer antigen, for instance, competes with unknown sample antigen for a limited number of antibody binding sites immobilized on a solid phase support. The tracer or detectible marker that is bound and quantitated is inversely proportional to the unknown analyte. In the two-site immunometric or "sandwich" architecture, for instance, high molecular mass analytes form a complex between immobilized antibody and tracer antibody with the signal being directly proportional to analyte concentration. These types of detectible markers (or tracers) which are employed with such immunodiagnostic assays can be varied and include radioisotopes, fluorescent materials, enzymes (which for purposes of the present invention are classified as catalysts for a detectible marker which is, for example, visually or spectrophotometrically perceivable), and the like. The term "label" is used herein to generically cover the aforementioned markers and catalysts (e.g., enzymes). One promising approach is to immobilize the antibody or antigen, as appropriate, in the particular type of assay desired on a waveguide material through which light can be appropriately directed to generate an evanescent wave adjacent the interface between the bulk solution containing the unknown to be measured and the waveguide surface. It is within this evanescent wave field that the detectible marker can be visually detected and measured.
Liposomes have been used as one component of prior art immunodiagnostic assays. In most cases, they have been used to entrap fluorescent dyes, enzymes, radioactive compounds, and the like inside the vesicles with either subsequent lysis or disruption of the vesicles to release the signal enhancing compounds. For example, U.S. Pat. No. 4,743,560 to R. L. Campbell describes a solid phase assay which features a liposome component which includes a detectible marker which is not visible.
U.S. Pat. No. 4,707,453 describes a solid phase assay in which vesicles are used which are formed, in part, from an amphiphilic chelating agent whereby a detectible metal marker can be complexed to the vesicle. The reference indicates that the detectible marker is external to the bilayer of the vesicle or sac in the sense of being both inside and outside the sac. The metal markers which are described for use in this patent are attached to the liposome substrate via chelating agents by means of a non-covalent bond which can dissociate.
A. Huang et al. in Journal of Immunological Methods, 46 (1981) 141-151, describes labeling of liposomes with ligands (i.e., antibodies) and fluorescent groups. This publication indicates that the immunoliposome labeling technique described therein was potentially very versatile without any specific description of an immunodiagnostic assay protocol or architecture for implementation. Also, the method for immobilizing proteins to vesicles which is described by Huang et al. requires modification of the protein by attachment of hydrophobic ligands prior to vesicle formation. When such a procedure is used, one runs the risk of precipitating the protein and, for example, of incorporating the protein incorrectly oriented on the vesicle bilayer as the vesicles are formed.
Another prior art reference which relates to vesicles and their use in an assay is U.S. Pat. No. 5,045,478 which, at Col. 4, lines 14-33, indicates that although the detectable marker used in such an assay is generally enclosed or encapsulated within the sac or vesicle, the vesicle "may be derivatized with the marker in a manner similar to derivatizing the sac with a ligand" (Col. 4, lines 17-18). This reference, however, clearly indicates a concern for "premature rupturing" (Col. 4, line 65) of such sacs and indicates that, in general, the marker is to be released from the sac (see, for example, Col. 5, lines 8-20).